PROJECT SUMMARY/ABSTRACT Cerebral aneurysms affect up to 5% of the population. When cerebral aneurysms rupture, they cause devastating subarachnoid hemorrhage with greater than 50% mortality. Unruptured aneurysms are problematic because little is known about which aneurysms will rupture or when. Currently, the only treatments that prevent rupture are prophylactic cranial or endovascular surgery which are risky. A gap in knowledge persists about the pathophysiology of aneurysm formation, growth, and rupture. The long-term goal is to improve understanding of the mediators and mechanisms of cerebral aneurysm formation and rupture, and thereby identify therapeutic targets for the development of novel therapies. A pill could prevent an unruptured aneurysm from rupturing obviating the need for risky surgery. Chemokine (C-X-C motif) ligand 1 (CXCL1) is a promising target in aneurysm pathophysiology. In preliminary studies, we show 1) endothelial cells at bifurcations and bifurcation aneurysms significantly express CXCL1; 2) CXCL1 is significantly expressed in mouse and human aneurysms; 3) CXCL1-blockade inhibits mouse aneurysm formation; and 4) CXCL1-blockade decreases vascular cell adhesion molecule 1 expression and inhibits neutrophil infiltration at mouse aneurysms. CXCL1 release is induced by interleukin 17 (IL17). In preliminary studies, we show 5) endothelial cells at bifurcations significantly express IL17A receptor; 6) IL17A is significantly expressed in mouse and human aneurysms; 7) IL17A- blockade significantly inhibits mouse aneurysm formation and rupture; and 8) IL17A-blockade increases expression of e-cadherin which forms tight junctions between endothelial cells, and that e-cadherin inhibition results in increased macrophage infiltration between endothelial cells. Further, we show 9) estrogen deficiency which is believed to contribute to aneurysm rupture, upregulates T helper 17 cells, increases IL17A release, and increases aneurysm rupture in female mice; and 10) L17A-blockade reverses the increased mouse aneurysm ruptures seen with estrogen deficiency. Our findings lead to our central hypothesis that the IL17- CXCL1 pathway induces aneurysm formation and rupture mediated by infiltrating macrophages and neutrophils. This Early Established Investigator R01 application will test our hypothesis with these three specific aims: 1) CXCL1 is necessary for aneurysm formation and rupture; 2) CXCL1-mediated aneurysm formation and rupture is induced by IL17; and 3) CXCL1-induced aneurysm formation and rupture is mediated by infiltrating macrophages and neutrophils. The proposal is conceptually innovative in that we will propose a novel link between IL17, IL8 and CXCL1, and neutrophils and macrophages and precisely define their roles as mediators in cerebral aneurysm formation and rupture. The approach will be innovative in that we will use novel hemodynamic flow models, novel mouse model of cerebral aneurysm rupture, and adeno-associated virus(AAV)-CRISPR/Cas9-guide(g)RNA vector technology. The significance of this work is the potential identification of novel targets with direct translational benefit.